Behavioral, metabolic, and lipidomic characterization of the 5xFADxTg30 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is associated with both extracellular amyloid-ß (Aß) plaques and intracellular tau-containing neurofibrillary tangles (NFT). We characterized the behavioral, metabolic and lipidomic phenotype of the 5xFADxTg30 mouse model which contains overexpression of both Aß and tau. Our results independently reproduce several phenotypic traits described previously for this model, while providing additional characterization. This model develops many aspects associated with AD including frailty, decreased survival, initiation of aspects of cognitive decline and alterations to specific lipid classes and molecular lipid species in the plasma and brain. Notably, some sex-specific differences exist in this model and motor impairment with aging in this model does compromise the utility of the model for some movement-based behavioral assessments of cognitive function. These findings provide a reference for individuals interested in using this model to understand the pathology associated with elevated Aß and tau or for testing potential therapeutics for the treatment of AD.

Urotensin II receptor antagonism confers vasoprotective effects in diabetes associated atherosclerosis: studies in humans and in a mouse model of diabetes.

AIMS/HYPOTHESIS: The small, highly conserved vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis. However, its effects in diabetes-associated atherosclerosis have not been assessed. METHODS: Endothelial cells were grown in normal- and high-glucose (5 and 25 mmol/l) media with and without UII (10⁻8 mol/l) and/or the UII receptor antagonist, SB-657510 (10⁻8 mol/l). Apoe knockout (KO) mice with or without streptozotocin-induced diabetes were treated with or without SB-657510 (30 mg kg⁻¹ day⁻¹; n = 20 per group) and followed for 20 weeks. Carotid endarterectomy specimens from diabetic and non-diabetic humans were also evaluated. RESULTS: In high (but not normal) glucose medium, UII significantly increased CCL2 (encodes macrophage chemoattractant protein 1 [MCP-1]) gene expression (human aortic endothelial cells) and increased monocyte adhesion (HUVECs). UII receptor antagonism in diabetic Apoe KO mice significantly attenuated diabetes-associated atherosclerosis and aortic staining for MCP-1, F4/80 (macrophage marker), cyclooxygenase-2, nitrotyrosine and UII. UII staining was significantly increased in carotid endarterectomies from diabetic compared with non-diabetic individuals, as was staining for MCP-1. CONCLUSIONS/INTERPRETATION: This is the first report to demonstrate that UII is increased in diabetes-associated atherosclerosis in humans and rodents. Diabetes-associated plaque development was attenuated by UII receptor antagonism in the experimental setting. Thus UII may represent a novel therapeutic target in the treatment of diabetes-associated atherosclerosis.

Delayed intervention with AGE inhibitors attenuates the progression of diabetes-accelerated atherosclerosis in diabetic apolipoprotein E knockout mice.

AIMS/HYPOTHESIS: Formation of AGEs is increased in the diabetic milieu, which contributes to accelerated atherogenesis. We studied whether delayed treatment with AGE-inhibiting compounds, alagebrium chloride and pyridoxamine dihydrochloride, affected established atherosclerosis in experimental diabetes in comparison with the angiotensin-converting enzyme inhibitor, quinapril. METHODS: Streptozotocin-induced diabetic male Apoe (-/-) mice (n = 24 per group) received, by oral gavage, from week 10 to 20 of diabetes: no treatment; alagebrium (1 mg kg(-1) day(-1)); pyridoxamine (1 g/l in drinking water); or quinapril (30 mg kg(-1) day(-1)). Atherosclerotic lesion area (en face analysis) was evaluated for all groups. RESULTS: Delayed intervention with alagebrium decreased plaque area in the diabetic Apoe (-/-) mice compared with untreated mice (total plaque area: alagebrium 10.6 ± 1.6%, untreated, 15.1 ± 1.5%, p < 0.05). This anti-atherosclerotic effect was comparable with that achieved with quinapril (quinapril 8.4 ± 1.4%, vs untreated, p < 0.05). Pyridoxamine also attenuated plaque development in diabetic mice (5.7 ± 1.2% vs untreated 11.9 ± 1.1%, p < 0.05). The anti-atherosclerotic effect conferred by alagebrium and quinapril was associated with a significant reduction in vascular oxidative stress and circulating AGEs and methylglyoxal, although preformed AGEs were not removed from the vascular wall with either delayed intervention. CONCLUSIONS/INTERPRETATION: Inhibition of AGE accumulation, using a delayed intervention with alagebrium or pyridoxamine, significantly attenuated the progression of established diabetes-associated atherosclerosis, similar to results obtained with quinapril. These findings provide further evidence that blockade of AGE-mediated pathways may present a novel therapy for the prevention of atherosclerosis in diabetes.

Large artery biomechanics and diastolic dysfunctionin patients with Type 2 diabetes.

AIMS: To comprehensively characterize large artery biomechanical properties and examine their relationship to cardiac function in patients with Type 2 diabetes mellitus (DM). METHODS: Fifty-five individuals with Type 2 DM were compared with 66 age- and sex-matched healthy control subjects. Arterial biomechanical properties were assessed by systemic arterial compliance (SAC; two-element Windkessel model), carotid-femoral pulse wave velocity (PWVc-f), femoral-dorsalis pedis pulse wave velocity (PWVf-d) and carotid augmentation index. Cardiac structure and function were assessed by echocardiography. RESULTS: Individuals with Type 2 DM had lower SAC and higher PWVc-f when compared with the healthy population. The PWVc-f was significantly lower than the PWVf-d in control individuals, but this difference was not evident in individuals with Type 2 DM due to higher PWVc-f. Augmentation index was similar in both groups, but the time to the first systolic inflection (time to reflection) was shorter in the individuals with Type 2 DM. The individuals with Type 2 DM had a greater prevalence of diastolic abnormalities when compared with the control group. Arterial stiffness indices, including SAC and pulse pressure, correlated with left ventricular filling pressure (defined as peak velocity during early diastolic filling divided by the velocity of movement of the mitral valve annulus in early diastole; r = -0.33 and 0.36 respectively. CONCLUSIONS: Patients with Type 2 DM on standard medication showed preferential stiffening of the large central arteries. However, carotid augmentation index was not different between the two groups and is therefore not a reliable indicator of large artery stiffening in this patient group. Diastolic dysfunction, present in a significant proportion of this population with Type 2 DM, was closely associated with arterial stiffening, suggesting a common aetiology.

Cell division autoantigen 1 plays a profibrotic role by modulating downstream signalling of TGF-beta in a murine diabetic model of atherosclerosis.

AIMS/HYPOTHESIS: Excess accumulation of vascular extracellular matrix (ECM) is an important pathological process in cardiovascular diseases including diabetes-associated atherosclerosis. We explored how a recently identified molecule, cell division autoantigen 1 (CDA1), influences the profibrotic TGF-beta pathway leading to vascular ECM accumulation. METHODS: Expression levels of genes encoding for CDA1, TGF-beta and connective tissue growth factor (CTGF) were examined in aorta from Apoe(-/-) mice with or without diabetes. We used retroviral and adenoviral constructs to knockdown or overexpress Tspyl2, the gene encoding CDA1, in mouse vascular smooth muscle cells (VSMCs) with or without TGF-beta treatment in order to demonstrate the role of CDA1 in TGF-beta signalling. RESULTS: In vivo studies indicated that the mRNA levels of CDA1-encoding gene Tspyl2 and protein levels of CDA1 were elevated in the aorta of diabetic Apoe(-/-) mice, accompanied by increased levels of Tgf-beta (also known as Tgfb1), Ctgf and ECM accumulation. In vitro studies in vascular cells showed that TGF-beta treatment rapidly increased CDA1 protein levels, which then amplified TGF-beta signalling leading to upregulation of ECM genes. Knockdown of CDA1-encoding gene Tspyl2 to reduce cellular CDA1 level markedly attenuated TGF-beta-stimulated MAD homologue 3 (drosophila; SMAD3) phosphorylation and transcriptional activities. CDA1 overproduction increased and Tspyl2 knockdown decreased expression of TGF-beta receptor type I, TbetarI (also known as Tgfbr1), but not TGF-beta receptor type II, TbetarII (also known as Tgfbr2), providing a mechanism for CDA1's action in modulating TGF-beta signalling. Knockdown of CDA1-encoding gene Tspyl2 also blocked the profibrotic effect of TGF-beta in VSMCs. CONCLUSIONS/INTERPRETATION: CDA1 plays an important role in vascular ECM accumulation by amplifying TGF-beta signalling. This is critical for the profibrotic effect of TGF-beta in the vasculature. CDA1 is therefore a potential target for attenuating vascular ECM accumulation caused by enhanced TGF-beta action, as seen in diabetic atherosclerosis.

Rapid potentiation of endothelium-dependent vasodilation by estradiol in postmenopausal women is mediated via cyclooxygenase 2.

Estrogens influence cardiovascular function through direct and indirect effects and via genomic and nongenomic mechanisms. The pathways underlying the nongenomic mechanisms are not completely understood. Estrogen-induced responses in vascular cells have been shown to influence prostaglandins and cyclooxygenase (COX), a key enzyme in the production of prostaglandins, with two isoforms, COX-1 and COX-2. We investigated the effects of prostaglandins on the acute potentiation by 17beta-estradiol (E) of acetylcholine (ACh)-mediated vasodilation in the cutaneous vasculature. Using a double-blind placebo-controlled design, we assessed skin blood flow in 32 healthy, postmenopausal women by laser Doppler velocimetry with direct current iontophoresis of ACh and sodium nitroprusside before and after 6-wk treatment periods with aspirin (a nonspecific COX-1 and COX-2 inhibitor), diclofenac (predominantly a COX-2 inhibitor, which also inhibits COX-1), celecoxib (a specific COX-2 inhibitor), given at anti-inflammatory doses, or placebo. Blood flux values before iontophoresis of ACh did not differ between the treatment groups or after E administration, excluding a direct cutaneous vasodilator effect of the treatments or of E. Acute E administration enhanced the response to ACh after aspirin, diclofenac, and placebo; however, this effect was completely abolished with celecoxib treatment (P < 0.05). E had no effect on sodium nitroprusside-mediated vasodilation after any of the treatments. We conclude that the COX-2 pathway plays a specific role in the rapid E-induced potentiation of cholinergic vasodilation in postmenopausal women.

The HMG-CoA reductase inhibitor rosuvastatin and the angiotensin receptor antagonist candesartan attenuate atherosclerosis in an apolipoprotein E-deficient mouse model of diabetes via effects on advanced glycation, oxidative stress and inflammation.

AIMS/HYPOTHESIS: We evaluated the anti-atherosclerotic effect of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, rosuvastatin, and the angiotensin II receptor blocker (ARB), candesartan, alone and in combination, in the streptozotocin-induced diabetic apolipoprotein E-deficient (Apoe (-/-)) mouse. METHODS: Control and streptozotocin-induced diabetic Apoe (-/-) mice received rosuvastatin (5 mg kg(-1) day(-1)), candesartan (2.5 mg kg(-1) day(-1)), dual therapy or no treatment for 20 weeks. Aortic plaque deposition was assessed by Sudan IV staining and subsequent visual quantification. The abundance of proteins was measured using immunohistochemistry. RESULTS: Diabetes was associated with a fourfold increase in total plaque area. Rosuvastatin attenuated plaque area in diabetic mice in the absence of lipid-lowering effects. The anti-atherosclerotic effect of rosuvastatin was comparable to that observed with candesartan. A similar beneficial effect was seen with dual therapy, although it was not superior to monotherapy. Rosuvastatin treatment was associated with attenuated accumulation of AGE and AGE receptor (RAGE) in plaques. Similar beneficial effects on markers of oxidative stress were seen with the ARB and statin. Candesartan was more effective at reducing macrophage accumulation and collagen I abundance in plaques compared with rosuvastatin. The combined effect of candesartan and rosuvastatin was superior in reducing macrophage infiltration, monocyte chemoattractant protein-1 level, vascular AGE accumulation and RAGE abundance in the vascular wall. Furthermore, the combination tended to be more effective in reducing smooth muscle cell infiltration and connective tissue growth factor abundance in plaques. CONCLUSIONS/INTERPRETATION: Rosuvastatin has direct anti-atherosclerotic effects in diabetic macrovascular disease. These effects are independent of effects on lipids and comparable to the effects observed with candesartan.

PPARs and diabetes-associated atherosclerosis.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors affecting the regulation of various genes relevant to the pathogenesis of diabetic complications. A number of drugs have been developed to act as agonists of the three PPARs. To date, PPAR isoforms that have been identified are the alpha, beta/delta, and gamma isosforms. Fenofibrate and gemfibrozil are two drugs that act as PPARalpha agonists and are currently in use in the clinical setting. Rosiglitazone is a PPARgamma agonist also in clinical use. These drugs have proved very useful in regulation of either glucose or lipid metabolism and consequently are used in patients with type 2 diabetes. Here, we will review the anti-atherosclerotic potential of PPAR agonists with particular emphasis on recent studies in an animal model of diabetes-associated atherosclerosis, the streptozotocin diabetic apolipoprotein E deficient mouse. These studies have shown both PPARalpha agonists, gemfibrozil and fenofibrate, confer anti-atherosclerotic effects, partly independent of their metabolic effects. Similar positive findings have also been detected in a dose-dependent manner with the PPARgamma agonist, rosiglitazone. The potential clinical implications of these findings are also discussed in view of the recently reported results of the PROACTIVE and FIELD clinical trials with the PPAR agonists rosiglitazone and fenofibrate respectively.

Gemfibrozil decreases atherosclerosis in experimental diabetes in association with a reduction in oxidative stress and inflammation.

AIMS/HYPOTHESIS: It is postulated that peroxisome proliferator-activated receptor alpha agonists confer cardiovascular benefits in diabetes, independently of their effects on lipid metabolism. We investigated putative mechanisms responsible for these anti-atherogenic effects in an in vivo model of diabetes-associated atherosclerosis. MATERIALS AND METHODS: Control and streptozotocin-induced diabetic apolipoprotein-deficient mice received gemfibrozil (100 mg kg(-1) day(-1)) or no treatment for 20 weeks. Aortic plaque deposition was assessed by Sudan IV staining and subsequent en face quantification. Superoxide production was measured using lucigenin-enhanced chemiluminescence. Markers of pathways including inflammation and oxidative stress were measured using real-time RT-PCR. RESULTS: No significant effect of gemfibrozil was observed on glycated haemoglobin, cholesterol or insulin in diabetic mice. Diabetes was associated with a three-fold increase in plaque area and a significant increase in NADPH-dependent superoxide compared with control mice. Gemfibrozil significantly attenuated plaque area and superoxide production in diabetic mice. In addition, gemfibrozil reduced the expression of the genes encoding the NADPH oxidase subunits p47phox, gp91phox and Rac-1. In addition, gemfibrozil reduced the expression of the genes encoding nuclear factor kappa B (NF-kappaB) subunit, p65, the NF-kappaB-dependent chemokine monocyte chemoattractant protein-1, and tissue factor. CONCLUSIONS/INTERPRETATIONS: This study demonstrates that gemfibrozil exerts anti-atherogenic actions, independently of changes in cholesterol and glucose metabolism. Such findings emphasise the possible usefulness of fibrates such as gemfibrozil in a setting of atherosclerosis even in the absence of dyslipidaemia.